Fig 1: IL-6/sIL-6R-EZH2 signalling modifies the DNA methylation of the CpG site (-170 bp) in the SP4 promoter region in HMPCs. (A) The 35 CpG sites in the SP4 promoter region (2 kb upstream of the ATG site). (B) The methylation percentage of the 35 CpG sites stimulated by IL-6/sIL-6R in HMPCs. (C, D) The methylation percentage of the 26th CpG site (-170 bp) in the SP4 promoter region in IL-6/sIL-6R, EZH2 overexpression and EZH2 inhibition conditions in HMPCs. The data are represented as the means + SDs, n = 3; * P < 0.05, ** P < 0.01.
Fig 2: IL-6/sIL-6R-EZH2 signalling enhanced VEGF secretion and the expression levels of EZH2, DNMT3B and SP4 in 5/6 nephrectomy rats and CKD patients. VEGF secretion was assessed in 5/6 nephrectomy rats (A) and CKD patients (B). (C-E) The mRNA expression levels of EZH2 (A), DNMT3B (B) and SP4 (C) in sham, 5/6Nx, 5/6Nx + PD, 5/6Nx + PD + IL-6/sIL-6R and 5/6Nx + PD + IL-6/sIL-6R + EZH2 rats. (F) Western blotting analysis showed the protein levels of EZH2, DNMT3B and SP4 in 5/6Nx rats with corresponding treatments in A-C. (G-I) The mRNA expression levels of EZH2, DNMT3B and SP4 in stage IV-V CKD patients with PD therapy. The data are represented as the means + SDs, n = 3; * P < 0.05, ** P < 0.01.
Fig 3: IL-6/sIL-6R-EZH2 signalling regulates the expression of DNMT3B and SP4 in HMPCs. The mRNA expression levels of EZH2 (A), DNMT3B (B) and SP4 (C) in HMPCs under IL-6/sIL-6R and EZH2 overexpression treatment for 6 h, 12 h or 24 h. (D) The protein expression levels of EZH2, DNMT3B and SP4 in HMPCs after 24 h of IL-6/sIL-6R, EZH2-OE and IL-6/sIL-6R + EZH2-OE treatment and the quantitative analysis of the western blotting results. The final concentration of IL-6/sIL-6R treatment was 100 ng/ml. NC, normal control. The data are represented as the means + SDs, n = 3; * P < 0.05, ** P < 0.01.
Fig 4: IL-6/sIL-6R-EZH2 signalling regulates the expression of DNMT3B and SP4 in HMPCs. The mRNA expression levels of EZH2 (A), DNMT3B (B) and SP4 (C) in HMPCs with IL-6/sIL-6R, IL-6/sIL-6R + EZH2 siRNA, IL-6/sIL-6R + DZNEP, IL-6/sIL-6R + GSK, IL-6/sIL-6R + DZNEP + EZH2 siRNA and IL-6/sIL-6R + GSK + EZH2 siRNA treatments for 6 h, 12 h or 24 h. (D) Western blot analysis showed the protein expression levels of EZH2, DNMT3B and SP4 in HMPCs with the corresponding treatments for 24 h. The final concentration of the IL-6/sIL-6R treatment was 100 ng/ml. The data are represented as the means + SDs, n = 3; * P < 0.05, ** P < 0.01.
Fig 5: EZH2 is involved in IL-6/sIL-6R signalling and regulates DNMT3B and SP4 expression in HMPCs. Immunofluorescence staining results show EZH2 (A), DNMT3B (B) and SP4 (C) expression in HMPCs under IL-6/sIL-6R and IL-6/sIL-6R + GSK stimulation.
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